The present invention is directed to a pyridine-soluble extract of a microorganism which, when combined with trehalose dimycolate (TDM) and an acetone precipitated by-product of endotoxic glycolipids extracted with chloroform-methanol (ACP), provides a pharmaceutical composition possessing anti-animal tumor properties.
Bacteria such as Corynebacterium parvum have been the subject of experimental work to isolate and characterize the component responsible for inducing inhibition of tumor growth [see, for example, Anti Tumor Activity and Lymphoreticular Stimulation Properties of Fractions Isolated from C. parvum; Cantrell, et al. Cancer Research 39, pgs. 3554-3563 (September, 1979]. Apart from anti-tumor activity, C. parvum has shown to be a potent stimulator of the lymphoreticular system resulting in undesirable increases in spleen and liver weights and blastogenesis. Applicant has discovered that a pyridine-soluble extract of a microorganism possesses potent anti-animal tumor properties without the undesirable toxic effects associated with the prior art products.
Trehalose dimycolates (TDM), may be obtained from organisms such as, for example, M. avium, M. phlei, M. tuberculosis (Strain H 37 RV and Ayoma B), M. bovis BCG, M. smegmatis, M. kansasii, Nocardia rubra, M. bovinis and Corynebacterium diphtheriae.
Bacteria such as M. avium are grown, harvested and then heat killed. The cell mass is then extracted with several solvents and then an active, solvent soluble, fraction is extracted. This extract is further purified by a series of solvent extractions to provide crude TDM (See Biologically Active Components from Mycobacterial Cell Walls. I. Isolation and Composition of Cell Wall Skeleton and Component P.sub.3 ; Azuma, et al., Journal of the National Cancer Institute, Volume 52, pgs. 95-101, 1974) incorporated herein by reference. As disclosed in Azuma et al., crude TDM may then be further purified by centrifugal microparticulate silica gel chromatography to give purified TDM.
An acetone precipitated by-product of endotoxic glycolipids extracted with chloroform-methanol (ACP) does not possess tumor-regressive properties when used alone or in combination with trehalose dimycolate. For a more complete discussion of ACP, its properties and methods of production, reference is made to Peptides as Requirement for Immunotherapy of the Guinea-Pig Line-10 Tumor with Endotoxins; Ribi, et al, Cancer Immunol. Immunother., Volume 7, pgs. 43-58 (1979) incorporated herein by reference. ACP can be prepared from any Enterobacteriaciae including, but not limited to, the following genera: Salmonella, Shigella, Escherichia, Brucella, Bordetella, Citrobacter, Pseudomnas, Pasturella, Neisseria, Proteus, Klebsiella, and Serratia.
The following species are typically employed: S. minnesota, S. typhimurium, B. pertussis, B. abortus, S. enteritidis, E. coli, S. typhi, S. marcescens, S. typhosa, Shigella flexni, and S. abortus equi.
It is, therefore, an object of the present invention to provide a pharmaceutical composition containing a pyridine-soluble extract of a microorganism in combination with trehalose dimycolate and an acetone-precipitated by-product of (ACP) endotoxic glycolipids extracted with chloroform-methanol.
It is another object of the invention to provide a method of treating animal tumors in warm blooded animals using the composition containing the pyridine-soluble extract of a microorganism, TDM and ACP.